Three forms of vitamin B12 have long been recognized to occur in biology, aquacobalamin/hydroxycobalamin, methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) (Golding, B. T. Chem. Brit. 1990, 950). (See Formula I). Methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) play crucial roles in the B12-dependent enzyme reactions and are frequently referred to as the B12 co-enzymes. Two known B12-dependent enzymes exist in humans: methionine synthase, which is methylcobalamin (MeCbl)-dependent, and methylmalonyl-coenzyme A mutase, which is adenosylcobalamin (AdoCbl)-dependent. (Dolphin, D. (ed). B12; John Wiley & Sons, Inc.: New York, USA, 1982; Banerjee, R. (ed.) Chemistry and Biochemistry of B12; JohnWiley & Sons, Inc.: New York, USA, 1999). In short, methionine synthase and methylmalonyl-CoA mutase require the vitamin B12 derivatives methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), respectively, for certain enzymatic reactions in the body. For example, in the MeCbl-dependent methionine synthase reaction, a methyl group is transferred from methyl-tetrahydrofolate (a metabolite of folate) to homocysteine (Hcy) via MeCbl to give methionine and tetrahydrofolate. This reaction results in the conversion of homocysteine, an amino acid found in humans which has destructive, oxidative properties, back to methionine. This reaction has received much attention in the medical literature in recent years, because its impairment can lead to elevated levels of homocysteine, which is associated with an increased risk of cardiovascular, cerebrovascular and peripheral vascular disease, and other pathological conditions which are discussed below.
Thiol derivatives of B12, thiolatocobalamins, were first identified in the 1960's, but have not attracted much attention until recently. They are characterized by having a cobalt-sulphur bond in the upper (beta) axial position. (See Formula I). Glutathionylcobalamin (GSCbl (or GluSCbl), a thiolatocobalamin) has been recently isolated from mammalian cells. A method for preparing glutathionylcobalamin is the subject of U.S. Pat. No. 7,030,105, the contents of which are incorporated herein by reference.
Glutathionylcobalamin (GSCbl) is an important cobalamin metabolite in mammals and is more active than other cobalamins in promoting methionine synthase activity in rabbit spleen extracts. It has been proposed that, in vivo, GSCbl (or a closely related thiolatocobalamin adduct) is a precursor of the two coenzyme forms of vitamin B12—AdoCbl and MeCbl. An alternative role for GSCbl was also recently proposed, in which the formation of GSCbl prevents B12 from being scavenged by xenobiotics.
The exact biochemical pathway(s) that lead to the incorporation of cobalamins into the B12-dependent enzymes have not yet been elucidated. It is known that thiolatocobalamins can be reduced by free thiols, yielding cob(l)alamin species, which can in turn be methylated by S-adenosylmethionine to form methylcobalamin. Whether this is an important biochemical pathway in humans needs further study.
A variety of thiolatocobalamins have been synthesized. Recently, simple synthetic methods have been reported for the preparation of three additional thiolatocobalamins—D,L-homocysteinyl-cobalamin (HcyCbl), the sodium salt of N-acetyl-L-cysteinylcobalamin (Na[NACCbl]), and 2-N-acetylamino-2-carbomethoxy-L-ethanethiolatocobalamin (NACMECbl). (Suarez-Moreira, E., Hannibal, L., Smith, C., Chavez, R., Jacobsen, D. W., and Brasch, N. E., Dalton Trans., in press. However GSCbl is the only thiolatocobalamin that has been isolated in mammals to date. Furthermore, prior to this synthesis, Na[NACCbl] and NACMECbl were not even reported to exist.
Formula I, below, depicts the structures of vitamin B12, including the two coenzyme forms of vitamin B12 and related B12 derivatives found in humans, all commonly referred to as the cobalamins.

The cobalamins belong to a family of compounds known as the corrinoids, which differ from one another in the specific nucleotide occupying the α axial site of the cobalt-corrin complex. The α (or lower) axial site is occupied by an intramolecularly-bound 5,6-dimethylbenzimidazole, and the β (or upper) axial site can be occupied by a variety of ligands. The various thiol ligand structures for the thiolatocobalamins mentioned herein are shown below.

It is known that vitamin B12 and its derivatives play key roles in human, animal and microbial metabolism. In humans, vitamin B12 helps maintain healthy nerve cells and red blood cells. It is also needed to produce DNA, the genetic material in all cells. (National Institutes of Health, Office of Dietary Supplements, Dietary Fact Sheet: Vitamin B12). Cobalamins (Cbls) are bound to protein in food, and hydrochloric acid in the stomach releases vitamin B12 from proteins during digestion. Once released, Cbls combine with a protein known as salivary haptocorrin (HC, also known as R-binder). Upon pancreatic proteolytic degradation of HC, Cbl is transferred in the duodenum to intrinsic factor (IF), which can then be absorbed by the GI tract. Cbl is transferred to transcobalamin (TC, TCII) within enterocytes. A substantial portion of TC-Cbl entering the portal vein after absorption is cleared by hepatocytes. Any free Cbl entering the circulation binds to either TC or HC.
Vitamin B12 deficiencies can occur in humans in a number of circumstances. Deficiencies can occur from malabsorption problems (damage to the GI tract lining, achlorhydria, inflammatory bowel conditions, infections, lack of intrinsic factor or other genetic anomalies), lack of a diet rich in vitamin B12, or the inability to utilize absorbed vitamin B12 and enzymatic or amino acid deficiencies. Certain drugs can also interfere with the absorption of vitamin B12.
Vitamin B12 deficiency can manifest in several different ways, including but not limited to anemias (including megaloblastic anemia also known as pernicious anemia), weakness, fatigue, weight loss, neurological changes, such as neuropathies (numbness and tingling), depression, confusion, and cognitive decline (such as loss of memory and dementia).
Vitamin B12, along with folate and vitamin B6, are involved in homocysteine metabolism. Homocysteine is a non-protein amino acid reversibly formed and secreted during human metabolism. Homocysteine is, however, a neurotoxin, and an abnormal increase in plasma homocysteine levels has been implicated in many pathological conditions, such as cardiovascular disease, neural tube defects, osteoporosis, stroke and other cerebrovascular disease, peripheral vascular disease, and certain forms of glaucoma and is now recognized in Alzheimer's disease. (Tchantchou, F., “Homocysteine metabolism and various consequences of folate deficiency”. J. Alzheimer's Dis. August 2006; Vol. 9, No. 4: 421-27). Homocysteine is eliminated from the body and is regulated by the transmethylation and transsulfuration pathways.
Homocysteine, among other reactive species, plays a key role in inducing oxidative stress. Oxidative stress can be defined as a harmful condition that occurs when there is an excess of free radicals, a decrease in antioxidants, or both. (E.g., Halliwell B. Introduction: Free Radicals and Human Disease—Trick or Treat? In: Thomas, C. E., Kalyanaraman, B. (ed.) Oxygen Radicals and the Disease Process. 1st ed. Amsterdam. Harwood Academic Publishers. 1997. pp. 1-14). Free radicals cause damage to cells by attacking their lipids, proteins and DNA components. A free radical is any species that contains one or more unpaired electrons, which makes it more reactive so that it can react with other species to form new free radicals. (Goodall, H. Oxidative stress: an overview.) It is this cycle that can lead to damage to cells in the body from prolonged exposure to free radicals.
The term reactive species is used to describe free radicals and other molecules that are themselves easily converted to free radicals or are powerful oxidizing agents. (Id.) Hydrogen peroxide is another example of a reactive species found intracellularly and extracellularly in humans.
It is known that a deficiency of vitamin B12, folate, or vitamin B6 may increase blood levels of homocysteine. Studies have shown that the reverse is also true. It was recently reported that vitamin B12 and folic acid supplements decreased homocysteine levels in subjects with vascular disease and in young adult women, with the most significant drop in homocysteine levels being seen when folic acid was taken alone (Bronstrup, A. et al. “Effects of folic acid and combinations of folic acid and vitamin B12 on plasma homocysteine concentrations in healthy, young women.” Am J Clin Nutr 1998; 68: 1104-10; Clarke, R. “Lowering blood homocysteine with folic acid based supplements. Brit Med J 1998; 316: 894-98). It has also been reported that a significant decrease in homocysteine levels occurred in older men and women who took a multivitamin/multimineral supplement for 8 weeks (McKay, D. et al. “Multivitamin/mineral Supplementation Improves Plasma B-Vitamin Status and Homocysteine Concentration in Healthy Older Adults Consuming a Folate-Fortified Diet.” J. Nutrition 200; 130: 309-96).
A question has been raised as to whether homocysteine levels correlate with actual disease, disease risk or are simply a marker reflecting an underlying process such as oxidative stress which is responsible for both high homocysteine levels and the development of disease. (Seshadri, S. “Elevated Plasma Homocysteine Levels: Risk Factor or Risk Marker for the Development of Dementia and Alzheimer's Disease”. J. Alzheimer's Dis. August 2006; Vol. 9, No. 4: 393-398.) Furthermore, McCaddon et al. note that these mechanisms are not necessarily mutually exclusive—for example, elevated homocysteine levels may perhaps be both a cause and consequence of oxidative stress (McCaddon et al. “Functional Vitamin B12 deficiency and Alzheimer's Disease. Neurology 2002; 58 (9): 1395-99).
It is well-accepted that many vitamin B12-related conditions, regardless of cause, can be easily (and reversibly) treated by administering vitamin B12 or its hydroxycobalamin derivative, either orally or by injection into muscle tissue. As suggested by the above studies, vitamin B12 may also play a role in conditions associated with oxidative stress by decreasing levels of homocysteine or other reactive species.
Thiolatocobalamins present useful therapeutic alternatives to vitamin B12 or hydroxycobalamin administration or supplementation. McCaddon and coworkers suggested that GSCbl and related thiolatocobalamins might be more effective than currently available pharmaceutical B12 forms (CNCbl and hydroxycobalamin) in treating of conditions associated with oxidative stress such as Alzheimer's disease (AD) and other neurological diseases (McCaddon, A., Regland, B., Hudson, P.; Davies, G. Neurol 2002; 58: 1395-1399). Numerous studies show that oxidative stress is an important neurodegenerative element in AD and several other neurological diseases. Glutathionylcobalamin is a naturally occurring intracellular form of cobalamin and is more readily absorbed and retained longer than cyanocobalamin. It has been proposed that, in vivo, GSCbl is an intermediate in the conversion of biologically inactive cyanocobalamin to the active coenzyme forms adenosylcobalamin and methylcobalamin. The reducing agent glutathione (GSH) is required for the formation of GSCbl, and is likely to be present in lower levels in AD patients as compared with healthy individuals due to oxidative stress. Thus, GSCbl has the potential to offer a valuable, direct source of cobalamin in therapeutic applications requiring administration of a vitamin B12 derivative. Furthermore, reduced glutathione levels are associated with a wide range of pathophysiological conditions, including liver failure, malignancies, HIV infection, pulmonary disease, and Parkinson's disease. The following list is for example purposes only and, although extensive, is not exhaustive: Acetaminophen poisoning, Attention Deficit Disorder, Autistic Spectrum Disorders, Addison's disease, aging, Acquired Immunodeficiency Syndrome, Amyotrophic lateral sclerosis, ankylosing spondylitis, arteriosclerosis, arthritis (rheumatoid), asthma, autoimmune disease, Behcet's disease, burns, cachexia, cancer, candida, cardiomyopathy, chronic fatigue syndrome, chronic obstructive pulmonary disease, chronic renal failure, colitis, coronary artery disease, cystic fibrosis, diabetes mellitus, Crohn's disease, Down's syndrome, eczema, emphysema, Epstein Barr viral syndrome, fibromyalgia, glaucoma, Goodpasture syndrome, Grave's disease, hypercholesterolaemia, herpes, viral/bacterial/fungal infections, inflammatory bowel disease, systemic lupus erythematosis, senile and diabetic macular degeneration, malnutrition, Meniere's disease, Multiple Sclerosis, Myasthenia Gravis, neurodegenerative diseases, nutritional disorders, pre-eclampsia, progeria, psoriasis, rheumatic fever, sarcoidosis, scleroderma, shingles, stroke, vasculitis and vitiligo.
McCaddon and Davies recently reported on observations concerning the co-administration of N-acetyl-L-cysteine (NAC, a glutathione precursor and potent antioxidant) with B vitamin supplements in cognitively impaired patients, all of whom had high serum homocysteine levels and two of whom had low reported glutathione levels. Improvements in agitation, alertness, and cognitive function were observed in these patients. (McCaddon, A. and Davies, G. “Co-administration of N-acetylcysteine, vitamin B12, and folate in cognitively impaired hyperhomocysteinaemic patients.” Int. J. Geriatr Psychiatry 2005; 20: 998-1000).
McCaddon also reported more recent observations concerning additional hyperhomocysteinanemic patients with cognitive impairment. The case reports demonstrate an apparent clinical efficacy of the addition of 600 mg N-acetyl-L-cysteine (NAC) to B12 and/or folate regimens. (McCaddon, A. “Homocysteine and cognitive impairment; a case series in a General Practice setting.” Nutrition Journal 2006; 5:6).
In view of the potential benefits reported with the use of glutathionylcobalamin, other thiolatocobalamins are also of interest. In particular, the novel compound Na[NACCbl] and other salts of NACCbl are of interest as a potential treatment or supplement, especially considering the above-noted observations associated with co-administrating hydroxycobalamin and N-acetyl-L-cysteine to Alzheimer's patients. There is an impetus to further test these novel, synthetic compounds for biological activity.
An understanding of the stability of thiolatocobalamins is essential if these compounds are to be used for treatment or supplemental applications. It is also important when exploring the biological relevance of these compounds. A range of thiolatocobalamins have been synthesized, some novel, and studies have been initiated on the stability and reactivity of these compounds as well. Interestingly, the stability of a specific thiolatocobalamin is very dependent on the thiol itself, and can vary over several orders of magnitude.
In efforts to reduce the damaging effects of oxidative stress and to establish the role of thiolatocobalamin treatment or supplementation in conditions associated with oxidative stress, there is a need to identify useful, stable and reactive thiolatocobalamin species. There is also a need not only for simple convenient methods of preparing thiolatocobalamins for use in human and animal studies, but also to develop test protocols that better define the role of oxidative stress (including the effects of reactive species such as homocysteine and hydrogen peroxide) in cell damage. Finally, there is a need to demonstrate the effects of naturally occurring and novel thiolatocobalamins on both healthy cells and those subjected to oxidative stress, including among other things increased homocysteine or H2O2 levels, in order to identify useful therapeutic applications for thiolatocobalamins.